The DNA-dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics involved in the DNA-damage response (DDR) and non-homologous end joining (NHEJ) double strand break (DSB) repair pathways. Various anti-cancer therapeutic strategies, including ionizing radiation (IR) impart their efficacy by inducing DNA DSBs. Both genetic and pharmacologic studies have demonstrated that modulating the DDR and DSB repair pathways has a profound impact on the efficacy of DNA damaging therapeutic agents supporting the premise of targeting DNA-PK in cancer therapy. Development of DNA-PK inhibitors thus far has focused entirely on targeting the DNA-PKcs active site, three of which are currently in early phase clinical trials. We have exploited the requirement for DNA-PK activation of binding to DNA termini via the Ku 70/80 heterodimer to identify small molecule Ku inhibitors that inhibit DNA-PK via a novel mechanism. Preliminary data show that Ku-inhibitors abrogate DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing therapeutics. We have also proven that the observed cellular effects are a function of direct on-target Ku inhibition. Based on the rigorous published and preliminary data we hypothesize that DNA-PK inhibition mediated by targeting Ku-DNA binding, will inhibit the DDR and NHEJ pathways resulting in sensitization of cancer cells to DNA damaging anti-cancer agents. To address this hypothesis, we propose three specific aims. In Aim 1 we will develop highly potent and selective DNA-PK inhibitors by targeting the Ku-DNA interaction. Having established nanomolar inhibitors, chemistry efforts will focus on optimizing the physicochemical and pharmacokinetic properties to increase cellular uptake and bioavailability while retaining excellent potency and selectivity. In Aim 2 we will determine the molecular mechanism of action (MOA) of Ku inhibitors and elucidate how chemical inhibition of Ku impacts the cellular DDR and repair pathways. In Aim 3 we will interrogate how modulation of DSB repair via HRR and DDR signaling due to Ku inhibition impacts anticancer efficacy alone and in combination therapy in clinically relevant models of lung and ovarian cancer. We will also assess the impact of common cancer genetic mutations in genome stability and maintenance pathways (including BRCA1 and BRCA2) towards exploiting synthetic lethal interactions to enhance drug and radiation efficacy. Completion of these studies will provide essential information for the continued discovery and development of novel Ku-targeted DNA-PK inhibitors. The impact of this research thus extends beyond the generation of new knowledge, reagents and models to provide new molecularly targeted treatment options for a wide array of cancers that are currently difficult to treat effectively.